Antibody binding microbial heparin binding motif to retard or prevent microbial biofilm formation on implanted medical devices

ABSTRACT

Methods and reagents for ameliorating biofilm formation on a surface of an indwelling or implanted device in a patient resulting in decreased virulence of microorganisms such as  Candida  species and/or  Staphylococcus  species.

This application is a Continuation-In-Part of co-pendingPCT/US2013/031499 filed Mar. 14, 2013, which claims priority to U.S.application Ser. No. 61/636,243 filed Apr. 20, 2012, each of which isexpressly incorporated by reference herein in its entirety.

A novel interaction between Candida albicans (C. albicans) surfaceproteins and heparin, a drug commonly used in patients at risk forcatheter-associated biofilms and candidemia, is disclosed with anantibody that reduced or prevented a microorganism surface protein frombinding to heparin, and reduced catheter-associated biofilm formation,where the reduction in biofilm formation can reduce the microorganism'svirulence in a patient, with reduction encompassing any level ofdecrease.

Heparin is a highly sulfated, non-branched, anionic disaccharidecomposed of uronic acid, predominantly iduronic acid, in 1,4 linkagewith glucosamine. Heparin is a known anticoagulant that is often infusedin indwelling catheters to prevent the blood flowing through thecatheter from clotting. Heparan sulfate is composed of the samedisaccharide but is less sulfated and has a more varied structure thanheparin. Heparan sulfate linked to core proteins, such as syndecans orglypicans, forms heparan sulfate proteoglycans (HSPGs). HSPGs are widelyexpressed on mammalian cell surfaces and in the extracellular matrix.Heparin binding motifs can bind to both heparin and heparan sulfate.

Several microbial proteins bind heparin and heparan sulfate, e.g., HIV-1gp120, CypA, and Tat; hepatitis C envelope glycoprotein E2; herpessimplex virus glycoproteins B, C, and D; dengue virus envelope protein;Plasmodium falciparum circumsporozoite (CS) protein, Listeriamonocytogenes ActA, and LcI of Legionella pneumophila (1-11). Suchmicrobial protein interactions with surface HSPGs typically promotebinding and entry of the microbial organism into the mammalian cell(except for HIV-1 gp120 and Tat), but the mechanisms by which individualmicrobial proteins recognize heparin or heparan sulfates are largelyundefined. Positively charged tripeptides containing Lys or Arg in theamino terminus of L. monocytogenes ActA (aa 40-230) and in L.pneumophila LcI (aa 69-349) were shared with tripeptides in a number ofheparin-binding mammalian proteins such as EGF-like growth factor andvonWillebrand factor, but the precise amino acids that mediated bindingto heparin were not defined for ActA or LcI (10-11).

Eukaryotic proteins that bind heparin express either linear orconformational heparin binding motifs (HBM). Linear heparin bindingmotifs are short, conserved peptides containing both basic (B) andhydropathic (X) amino acids in specific patterns first identified byCardin (XBBXBX) and Weintraub (XBBBXXBX) and subsequently expanded bySobel (XBBBXXBBBXXBBX) (12-14). Molecular modeling studies suggestedthat basic amino acids such as lysine, arginine, and histidine werecritical for interaction with anionic sulfate or carboxylate groups inheparin through electrostatic and hydrogen bonds. For example,substituting alanine for basic amino acids in linear HBM in themorphogen sonic hedgehog abolished binding to heparin (15). However,because many heparin-binding proteins failed to exhibit linear motifs,the concept of spatial orientation of basic residues was propounded(16). The CPC clip motif, a structural signature in which two cationicresidues surround one polar residue, was conserved in eukaryotic heparinbinding proteins, as well as in vaccinia complement protein andpapillomavirus 18 capsid protein (17)

Because of its propensity to form biofilms in implanted medical devicessuch as indwelling central venous catheters, Candida albicans (C.albicans) is a leading pathogen in infections of indwelling catheters. Abiofilm is a multilayered structure of microbes embedded in apolysaccharide matrix that forms on central venous catheters as well asimplanted prosthetic joints, contact lenses, and other such medicaldevices (18, 19). A biofilm is undesirable because antibiotics and hostdefenses cannot penetrate it, thus a biofilm prevents elimination ofmicroorganisms that have attached or adhered to a surface of animplanted medical device. For example, because of the high flow-throughin a central venous catheter, sessile projections from a biofilm breakoff and are carried into the bloodstream to cause infection that can becarried by the blood to other sites in the body.

Heparin is a known anticoagulant, and catheters are often infused withheparin to prevent blood flowing through the catheter from clotting(20). Because heparin inhibited attachment of C. albicans toextracellular matrix proteins (21), the inventors hypothesized that C.albicans interacted with heparin through linear heparin binding motifs(HBMs): conserved sequences of basic (Arg, Lys, H is) and hydropathicamino acids in prescribed patterns.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the internal lumen of an indwellingcatheter.

FIGS. 2A-E demonstrate lack of an effect by heparin on wild type Candidaalbicans (C. albicans) planktonic growth and morphology.

FIGS. 3A-D demonstrate C. albicans binding to heparin and heparinanalogs in vitro.

FIGS. 4A-C show that two lysine residues in linear heparin binding Motif1 in C. albicans Int1 mediate binding to heparin, as measured by anELISA assay in vitro. FIG. 4A discloses SEQ ID NOS 1, 40, 48, 2, 3, 52and 53, respectively, in order of appearance. FIG. 4B discloses SEQ IDNO: 2. FIG. 4C discloses SEQ ID NOS 52-53, respectively, in order ofappearance.

FIG. 5 compares pre-immune rabbit IgG with affinity-purified IgG raisedagainst the sequence HKQEKQKKHQIHKV (SEQ ID NO:4) for ability to inhibitbinding of C. albicans to heparin.

FIGS. 6A-C show consequences of heparin binding in vitro. FIG. 6Adiscloses SEQ ID NO: 65. FIG. 6B discloses SEQ ID NO: 66. FIG. 6Cdiscloses SEQ ID NO: 67.

FIGS. 7A-B show consequences of heparin binding in vitro.

FIGS. 8A-F demonstrate that heparin binding motifs contribute to biofilmformation in vivo.

FIG. 9 demonstrates that affinity-purified IgG raised against thesequence HKQEKQKKHQIHKV (SEQ ID NO:4) substantially inhibited biofilmproduction in jugular venous catheters in rats. FIG. 9 discloses SEQ IDNO: 6.

FIG. 10 schematically illustrates the internal lumen of an indwellingcatheter in the presence of antibody against heparin binding motifs oncell wall proteins of C. albicans.

FIG. 11 shows Anti-KKHQ specific binding to Candida species.

FIGS. 12A-C show Anti-KKHQ effect on biofilm progression.

Thirty-four C. albicans surface proteins encoding at least one linearheparin binding motif (HBM) were identified from a sequence-basedsearch. Twelve of these 34 proteins flanked recently describedtranscriptionally active regions that are involved in biofilmproduction. Thirty-three of these 34 proteins are known targets ofmaster biofilm regulators (22).

C. albicans binding to 2.5 units (12.5 μg) solid-phase heparin in anELISA assay was significantly decreased by desulfation of heparin at the2-O or 2-N groups, and by preincubation of C. albicans with heparin. Theprotein Int1 contained the largest number of HBMs; Int1 is a surfaceprotein that is involved in adhesion, filamentation, and antigenicrecognition. In the Int1 sequence ₈₀₄QKKHQIHK (SEQ ID NO: 1), alaninemutation of lysine residues at positions 805/806 significantly reducedbinding of the Int1 mutant to heparin. Rabbit IgG recognizing thepolypeptide ₇₉₉HKQEKQKKHQIHKV₈₁₂ (SEQ ID NO: 4) inhibited C. albicansbinding to heparin by 19%; pre-immune rabbit IgG had no effect.

Consequences of heparin binding in vitro included removal of C. albicanssurface antigens such as Eno1, Pgk1, Tdh3, and Ssa1/2, which themselvescontain putative HBMs; impairment of histatin-mediated killing; andmodulation of gene expression. In vivo, substitution of alanine residuesfor lysines at positions 805/806 in ₈₀₄QKKHQIHK (SEQ ID NO: 1) markedlyattenuated biofilm formation in central venous catheters in rats. Inaddition, pre-incubation of C. albicans with rabbit IgG recognizing thepolypeptide ₇₉₉HKQEKQKKHQIHKV₈₁₂ (SEQ ID NO: 4) inhibited biofilmformation in vivo; pre-immune IgG had no inhibitory effect. Theseresults identify linear HBM in C. albicans surface proteins,characterize specific lysine residues that mediate heparin binding, anddemonstrate relevance for innate and adaptive immunity in vitro andbiofilm formation in vivo.

The inventive method and composition ameliorated this undesirablesituation by using antibodies against a specific heparin-binding motifthat is expressed by surface proteins on C. albicans; othermicroorganisms also express similar heparin binding motifs. Withoutbeing held to a single theory, the disclosed antibodies reduce orprevent the undesirable biofilm from forming on the surface of a medicaldevice, particularly a plastic device, implanted in a patient.Microorganisms include bacteria, yeast, fungi, etc. as known to oneskilled in the art. Medical devices include those implanted orimplantable in a patient, particularly plastic medical devices,including but not limited to catheters and central lines but excludingcurrent non-plastic implanted joints, pacemakers, pacemaker wires, andspinal rods.

In one embodiment of the inventive method, antibodies were producedagainst a linear heparin binding motif that was expressed by a surfaceprotein of Candida albicans (C. albicans). Related heparin bindingmotifs occur in surface proteins from Staphylococcus epidermidis (S.epidermidis) and Staphylococcus aureus (S. aureus).

Linear heparin binding motifs are sequential amino acids that conform toone of three consensus motifs (Cardin, Sobel, or Weintraub motifs); theyhave been identified in multiple mammalian proteins that bind heparin.While conformational heparin binding motifs also occur in some proteins,they must be identified by crystal structure of the protein. Antibodiesto these linear heparin binding motifs inhibited C. albicans fromadhering to heparin bound to a plastic surface (a microtiter plate).

Of the six million patients who have a central line implanted each year,more than 75,000 will develop bloodstream infections (23). Centralline-associated blood stream infections (CLABSI) are a major source ofhospital-acquired infection; over 43,000 CLABSI occurred among patientshospitalized in U.S. intensive care units (ICUs) in 2001, which is 3.2infections for every 1000 line days. With a protocol for their sterileinsertion and daily maintenance, CLABSI in ICUs dropped to 18,000 in2009, but even with these improvements in ICUs procedures, more than23,000 CLABSI occurred in patients on inpatient wards and 37,000 CLABSIoccurred in outpatient hemodialysis patients in 2009. Even with a 50%reduction in CLABSI in ICU patients, the annual cost of these infectionsacross the U.S. is more than $13 billion (23)

Staphylococcus epidermidis is the most common cause of CLABSI in centrallines, with gram-negative rods second, S. aureus third; and Candidaspecies fourth (24). In peripherally inserted central catheters (PICC),i.e., central lines inserted through peripheral veins in the arm,Candida species are the second most common cause of line infection (25).Candida spp. are equivalent to S. aureus in infections in central linesin the ambulatory setting (26). Among Candida spp, C. albicans is themost common cause of CLABSI (24).

When Candida species infect a central venous catheter, the organismenters the bloodstream to cause candidemia. Hosts at highest risk ofcandidemia, include burn patients and patients on a pump during coronaryartery bypass procedures (27). Neutropenic oncology patients, prematurenewborns, and patients with major abdominal surgery such as intestinalresections are also high-risk patients with 8- to 16-fold the number ofinfections of other patient groups (27).

A major risk factor in candidemia is the presence of central lines(28-33), whose lumens are a site for formation of microbial biofilmsthat are effectively shielded from antimicrobial agents and hostdefenses. A second commonality is the use of heparin as an anticoagulantin most catheters. The amount of heparin is often considerable; e.g., apremature newborn in a neonatal intensive care unit may receive 150units/kg/day, compared to 2.8 units/kg for a 70 kg adult receiving a 100unit flush twice a day (20).

The ability to bind heparin is a unifying feature among S. epidermidis,S. aureus, and, Candida albicans (34-36), three of the leading causes ofcatheter-associated bloodstream infections. In experiments with S.aureus, high concentrations of heparin have been shown to increase S.aureus biofilm formation in vitro (37).

A first step in biofilm formation is microorganism adhesion or adherenceto a surface of the implanted device, such as a catheter (38). In FIG.1, negatively charged heparin molecules (circles) injected into thecatheter lumen bind to the positively charged catheter surface, andheparin sulfate moieties (curved lines) are exposed on vascularendothelium lining catheter lumens. Linear heparin binding motifspresent on the surface of microorganisms, such as C. albicans, interacteither with heparin (A) or with heparin sulfate (B) and enable theorganism to attach to the inside of the catheter. As the attachedmicroorganisms replicate, they form the multilayered structure of abiofilm (C) and secrete the polysaccharide mortar-like matrix that holdsthe biofilm together. The inventors hypothesize that interactionsbetween heparin and surface proteins on microorganisms such as C.albicans facilitate biofilm formation, and subsequent infection of thebloodstream when projections from biofilm break off into the catheterlumen and enter the bloodstream.

Heparin is associated with central line infections, particularly thosedue to Candida spp. In a randomized trial of 260 central lines, 128 werecoated with chlorhexidine and 132 were coated with heparin; all Candidacolonization and all candidemias occurred in patients whose centrallines were coated with heparin (39). A second study in renal dialysispatients showed that those patients who received 5000 units of heparinin the middle of dialysis had considerably more catheter loss due toinfection compared to patients not treated with heparin. Althoughstaphylococcal infections were most common in this patient group,candidemia also occurred (40).

One mechanism by which heparin interacts with proteins is through linearheparin binding motifs, conserved sequences of basic amino acids such aslysine, arginine, and histidine interspersed with hydropathic aminoacids (14). Microbial heparin-binding proteins are identified by shortconsensus motifs of basic (B) and hydropathic (X) amino acids, asdefined by Cardin [XBBXBX], Weintraub [XBBBXXBX], or Sobel[XBBBXXBBBXBBX].

In the invention, a computer-based algorithm examined all 400 C.albicans surface and cell wall proteins for heparin binding motifs(HBM). Of the 6,000 known Candida albicans proteins, about 400 arelocalized to the cell wall of yeast or hyphae, depending upon theextraction technique (41). A sequence-based search identified putativeCardin, Sobel, or Weintraub motifs in 159 C. albicans proteins. Table 1shows HBM in cell surface proteins expressed by C. albicans. The 34proteins in Table 1 include only those that have the attribution “cellsurface”, “fungal cell wall”, “yeast cell wall”, or “hyphal cell wall”as the cellular component in the Candida Genome Database, where*=Weintraub motif; #=Cardin motif; =Identified by molecular modelingstudies; A=“cell wall” in protein description (as of January 2011);obtained from Candida Genome Database (CGD); B=“cell wall” in GeneOntology (GO) annotation (as of January 2011); obtained from CGD; C=cellwall proteins as reviewed by Alberti-Segui (42) and D=cell wall proteinsas reviewed by Chaffin (41).

The following procedure was used to identify a set of cell wall proteinsin C. albicans with HBM. The first step combined evidence to identifypossible cell wall proteins in C. albicans. Specifically, using “cellwall” as keywords, 154 putative cell wall proteins were identified fromprotein descriptions, and another 245 unique cell wall proteins wereidentified from Gene Ontology functional annotations from Candida GenomeDatabase (www.candidagenome.org; Assembly 21). Also included were 125non-redundant cell wall proteins identified by Alberti-Segui (50) andanother 174 reported by Chaffin (22) and mapped annotations to Assembly21. The second step screened for consensus HBM in protein ORF sequencesof these cell wall proteins from Assembly 21. Three types of consensusmotifs were included: Weintraub (XBBBXXBX), Sobel (XBBBXXBBBXXBBX), andCardin (XBBXBX) motifs, where B is a basic amino acid and X is ahydropathic amino acid. In the algorithm, basic amino acids are H, K,and R. Hydropathic amino acids are W, F, Y, L, I, C, M, G, V, S, T, A,N, P, and Q. A total of 159 cell wall proteins were identified that havepossible HBM, and some have multiple motifs. The 159 proteins that metthe search criteria were then manually curated to select only thoseproteins whose cell wall localization was confirmed by manual orcomputational methods in the Candida Genome Database.

Proteins with GPI anchoring sequences within these 159 cell wallproteins with HBM were further identified. Three Web servers were usedto perform GPI anchoring sequence prediction: SignalP (43), GPI-SOM(44), and PredGPI (45). Combined results of these three predictionmethods indicated that 15 out of these 159 proteins had GPI anchoringsequences, four of which overlapped with reviewed GPI anchoring proteins(46).

The algorithm identified 34 cell wall proteins with potential linearheparin binding sequences that matched the Cardin, Weintraub, and Sobelmotifs. The 34 identified cell wall proteins were Ahp1, Als7, Atcl,Bud2, Cat1, Cef3, Chs1, Crh12, Dot4, Not5, Pdi1, Pga4, Pgk1, Phr3, Rbt1,Rps6A, Sam2, Srb1, Ssa2, Ssb1, Tdh3, Eft2, Eno1, Gap1, Gph1, Gpm2,Hem13, Hsp104, Hsp70 (also called Ssa1), Ino1, Int1, Ipp1, Ugp1, Xyl2.Three of these proteins, Als7, Pga4, and Rbt1, have GPI anchors. Thegenes encoding eleven of these proteins, Als7, Cat1, Dot4, Eno1, Gph1,Ino1, Rbt1, Sam2, Srb1, Ssa2, and Ugp1, are located in newly definedtranscriptionally active regions that are critically involved in biofilmformation (22). Moreover, many of these proteins are known to beregulated by at least one of six master biofilm regulators; by RNA-seq,where RNA-seq is defined as the use of high-throughput sequencingtechniques to sequence cDNA in order to get sequence information aboutthe transcriptome, the sample's RNA content; CAT1, GAP1, GPH1, andHSP104 are up-regulated in biofilm formation, while CHS1, EN01, GPM2,HEM13, INO1, IPP1, PGK1, RPS6A, SAM2, SRB1, SSA2, SSB1, and TDH3 aredown-regulated (22)

Given the presence of putative HBM among C. albicans surface proteinsand the possible interactions with heparin in central venous cathetersor with heparin sulfate proteoglycans expressed on host tissues such asvascular endothelium, the biochemical determinants and immunologicconsequences of this interaction were defined. The protein Int1 (SEQ IDNO: 5) (accession number P53705.2; GI 187608862) had the highest numberof HBM (five). Int1 (SEQ ID NO: 5) spans 1711 amino acids and islocalized to the cell wall of the bud neck in C. albicans. It mediatesadhesion, hyphal formation, and virulence, defined as the ability tocause disease such as bloodstream infection (47). Using the same searchtechnique, HBM were also detected in surface proteins of S. epidermidisand S. aureus.

TABLE 1  Gene ID # of (Assembly 21) Protein Ref. motifs Motif 1 Motif 2Motif 3 Motif 4 Motif 5 orf19.4257 Int1 C 5 ₈₀₄QKKHQIHK^(‡)₁₃₈₃THKGRF^(#) ₁₅₃₀MKRGKP^(#) ₁₅₉₃FKKRFFKL* ₁₆₁₂SHKTRA^(#)(SEQ ID NO: 1) (SEQ ID NO: 40) (SEQ ID NO: 48) (SEQ ID (SEQ ID NO: 2)NO: 3) orf19.1738 Ugp1 B, D 3 ₁₇₆SHRIRV^(#) ₃₁₀IKKFKY^(#) ₃₆₈IRHFKG^(#)(SEQ ID NO: 7) (SEQ ID NO: 41) (SEQ ID NO: 49) orf19.3370 Dot4 B 3₄₄₂NKKGKS^(#) ₅₁₉CHKCHN^(#) ₆₃₅FKRFKF^(#) (SEQ ID NO: 8) (SEQ ID NO: 42)(SEQ ID NO: 50) orf19.3651 Pgk1 A, B, D 3 ₁₃₆GKKVKA^(#) ₁₄₆VKKFRQ^(#)₁₆₈AHRAHS^(#) (SEQ ID NO: 9) (SEQ ID NO: 43) (SEQ ID NO: 51) orf19.4660Rps6A B, D 2 ₁₈₅QRKRALKA* ₁₉₂AKKVKN^(#) (SEQ ID NO: 10) (SEQ ID NO: 44)orf19.5107 Not5 B 2 ₁₁₄QKRSRF^(#) ₃₃₃VKKLKP^(#) (SEQ ID NO: 11)(SEQ ID NO: 45) orf19.5130 Pdi1 D 2 ₂₁₀NKKFKN^(#) ₃₀₁GKKYRG^(#)(SEQ ID NO: 12) (SEQ ID NO: 46) orf19.6387 Hsp104 D 2 ₅₃VKRARY^(#)₁₉₉ARRSKS^(#) (SEQ ID NO: 13) (SEQ ID NO: 47) orf19.1065 Ssa2 B, D 1₂₅₈LRRLRT^(#) (SEQ ID NO: 14) orf19.1067 Gpm2 B 1 ₄₅IKKNHL^(#)(SEQ ID NO: 15) orf19.1327 Rbt1 A, B, C 1 ₁₂₁GKKVKQ^(#) (SEQ ID NO: 16)orf19.2762 Ahp1 B, D 1 ₁₇₁LKRIHN^(#) (SEQ ID NO: 17) orf19.2803 Hem13B, D 1 ₂₅₇IRRGRY^(#) (SEQ ID NO: 18) orf19.3590 Ipp1 B, D 1 ₇₄TKKGKL^(#)(SEQ ID NO: 19) orf19.377 Phr3 B, C 1 ₁₁₅PHHHLNRY* (SEQ ID NO: 20)orf19.395 Eno1 B, D 1 ₁₄₁AKKGKF^(#) (SEQ ID NO: 21) orf19.3966 Crh12A, B, C 1 ₄₂₀TKHIHN^(#) (SEQ ID NO: 22) orf19.4035 Pga4 B, C, D 1₂₅₉AKRPRP^(#) (SEQ ID NO: 23) orf19.4152 Cef3 B 1 ₆₂₃LRKYKG^(#)(SEQ ID NO: 24) orf19.4304 Gap1 B 1 ₇₂QRKLKT^(#) (SEQ ID NO: 25)orf19.4980 Ssa1 B, D 1 ₂₅₉LRRLRT^(#) (SEQ ID NO: 26) orf19.5188 Chs1B, C 1 ₁₂₆PKRQKT^(#) (SEQ ID NO: 27) orf19.5788 Eft2 B, D 1₅₈₁NKHNRI^(#) (SEQ ID NO: 28) orf19.6190 Srb1 B, D 1 ₁₂₃FHKAHG^(#)(SEQ ID NO: 29) orf19.6214 Atc1 A, B 1 ₉₅₉PKRVKV^(#) (SEQ ID NO: 30)orf19.6229 Cat1 B 1 ₈₂GKKTRI^(#) (SEQ ID NO: 31) orf19.6367 Ssb1 B, D 1₂₆₃LRRLRT^(#) (SEQ ID NO: 32) orf19.657 Sam2 B, D 1 ₃₈₀PKKLKF^(#)(SEQ ID NO: 33) orf19.6814 Tdh3 B, D 1 ₇₀GHKIKV^(#) (SEQ ID NO: 34)orf19.7021 Gph1 B, D 1 ₆₅₆TKHHIPKA* (SEQ ID NO: 35) orf19.7400 Als7 B, C1 ₁₄₈₂SKRNKN^(#) (SEQ ID NO: 36) orf19.7585 Ino1 B, D 1 ₁₆₁MKRAKV^(#)(SEQ ID NO: 37) orf19.7676 Xyl2 A, B, D 1 ₃₃₀THRFKF^(#) (SEQ ID NO: 38)orf19.940 Bud2 B 1 ₇₁₆LRKGKS^(#) (SEQ ID NO: 39)

Heparin effect on Candida albicans growth and morphology was determined.A single colony of BWP17 wt was inoculated into 3 ml YPD medium andincubated overnight at 30° C. with shaking at 225 rpm. Overnightcultures were diluted to an OD₆₀₀ of 0.1 in 5 ml of YPD (yeast) orRPMI-HEPES (hyphae) in a 50 ml conical polypropylene tube and incubatedin the presence or absence of 100 units/ml preservative-freepharmaceutical heparin at 30° C. for six hours. Cells (1 ml) were fixedwith an equal volume of 4% formaldehyde in FACS buffer at 4° C. for onehour, then pelleted (7,000 rpm for 3 minutes) and washed with PBS. Afterreconstitution in 0.5 ml PBS, filipin (50 mg/ml stock solution in DMSO)was added to a final concentration of 100 μg/ml and incubated with cellsat room temperature for five minutes. Cells were pelleted, washed withPBS and mounted on a slide using Fluoromount G. For calcofluor whitestaining, after six hours incubation, 1 μl of 1 mg/ml stock solution ofcalcofluor white in 0.1 N NaOH was added to 100 μl cells to a finalcalcofluor white concentration of 10 μg/ml.

Microscopy was performed by imaging filipin, PKH26, DAPI, andheparin-Alexa Fluor 488 on a Nikon Ti-E inverted microscope with a100×CFI APO oil NA 1.49 objective. Filipin was excited with a PriorLumen 200 metal halide light source set at 10% light output. This lightwas further attenuated by ND4 and ND8 neutral density filters in seriesto reduce light output to approximately 3% of output from the liquidlight guide. The filters used for imaging were EX 360/40, dichroic 400nm Ip, EM 460/50. Images were acquired with an Andor iXon emccd camera.Exposure times were 391 ms. Excitation of PKH26/DAPI/Heparin-Alexa Fluor488 triple staining was accomplished with a Nikon A1R si laser scanningconfocal. DAPI was imaged with 405 nm excitation and a 450/50 filterwith a laser power of 16.3 and a photomultiplier tube (pmt) voltage of92. Alexa Fluor 488 was imaged with 488 nm excitation from an Argon-ionlaser and a 525/50 filter with a laser power of 6.0 and a pmt voltage of84. PKH26 was imaged with 561 nm excitation and a 595/50 filter with alaser power of 13.2 and a pmt voltage of 108. Gains were kept below 110to eliminate contribution from C. albicans autofluorescence; lack ofautofluorescence was confirmed by comparison to a control sample withoutheparin-Alexa Fluor 488. Images were processed with Nikon NIS-ElementsAR 4.11.00 64-bit software. Calcofluor white slides were examined usinga Zeiss Axiovert 200M fluorescent microscope equipped with aPlan-Apochromat 63×/1.40 oil objective lens with 1.6× optivar and DAPIfilter at 350 nm excitation and 460 nm emission. Images were capturedusing a Zeiss Axiocam color camera and processed with Axiovision version4.8.2.

Flow cytometry was performed by inoculating a single colony of BWP17 wtinto 3 ml YPD medium and incubating overnight at 30° C. with shaking at225 rpm. Overnight cultures were diluted to OD₆₀₀ of 0.1 in 5 ml of YPDor RPMI-HEPES and incubated in the presence or absence of 100 units/mlpreservative-free pharmaceutical heparin at 30° C. for one hour. One mlaliquots of each mixture were removed, pelleted (7,000 rpm for 3 min),washed twice with PBS, and reconstituted in 1 ml PBS. Flow cytometryanalysis was performed using the Imagestream^(X) flow cytometer (Amnis,Seattle Wash.) equipped with a 405 nm, 488 nm, 653 nm, laser andmulti-mag function. The 40× magnification, 10 mm/sec flow rate, and 488nm laser were used to collect SSC and Brightfield parameters. The flowcell allows particles up to 100 μm wide (height unlimited) to becollected in the instrument. INSPIRE (v.6.0) software was used toacquire events. Software analysis using IDEAS (v5.0) identified percenthyphae using aspect ratio, height and width features using the sidescatter (Channel 6) and brightfield (Channel 1) parameters. Objects wereselected within each file and tagged to use as identification of truthsets within the population.

For growth curves in the presence or absence of soluble heparin, asingle colony of BWP17 wt was inoculated into 3 ml YPD medium andincubated overnight at 30° C. with shaking at 225 rpm. Overnightcultures were diluted to an OD₆₀₀ of 0.1 in 5 ml of YPD, RPMI-MOPS,RPMI-HEPES or CSM in a 50 ml conical polypropylene tube.Preservative-free pharmaceutical heparin (1000 units/ml) was added toyield a final concentration of 100 units/ml in the heparin-treatedsamples. Cultures were grown at 30° C. with shaking and OD₆₀₀ measuredperiodically. Doubling times (t_(d)) were calculated based on theequation t_(d)=ln 2/μ (μ=specific growth rate (48). One hundred units/mlheparin, the concentration recommended to prevent clotting of centralvenous catheters (20), had no effect on doubling times of planktonicyeast cells grown in YPD, RPMI-HEPES, RPMI-MOPS or CSM, as shown inFIGS. 2A and 2B.

FIGS. 2A-E demonstrate lack of an effect by heparin on wild type Candidaalbicans (C. albicans) planktonic growth and morphology.

FIG. 2A shows that growth curves in the presence (open circles) orabsence (closed diamonds) of soluble heparin were identical.

FIG. 2B shows that doubling times in various media at 30° C. in theabsence and presence of heparin (100 units/ml) were identical.

FIG. 2C shows that percent hyphae as determined by flow cytometry didnot differ after incubation with or without heparin (100 units/ml) underconditions that favor yeast (YPD, 30° C.) or hyphae (RPMI-HEPES, 37°C.).

FIGS. 2D-E show that the integrity of membrane sterols identified withfilipin (FIG. 2D) and the location of septin rings identified withcalcofluor white staining (FIG. 2E) were identical between untreated andheparin-treated organisms. Thus, concentrations of heparin commonly usedto maintain patency of central venous catheters (100 units/ml) did notlead to readily observable differences in growth or cellulararchitecture of the organism.

Binding of heparin by C. albicans was examined in vitro. Heparin waslabeled with Alexa Fluor 488 by a method modified from Osmond (49).Briefly, 5 mg heparin (Sigma) was dissolved in 0.5 ml MES buffer (0.1M2-(N-morpholino)ethanesulfonic acid hydrate, pH 4.5, Sigma), then mixedwith a solution of 1 mg Alexa Fluor 488 hydrazide (Life Technologies) in0.4 ml MES buffer. After adding 0.2 ml of EDC solution (15 mg1-ethyl-3-(3-dimethylaminopropyl) carbo-diimide/ml water, ThermoScientific), the mixture was stirred at room temperature for 30 min. Asecond 0.2 ml aliquot of EDC solution was added and the mixture stirredat room temperature for an additional 30 min. After adding 1.3 ml NaOAc(1 M, pH 4.8) and stirring at room temperature for one hour,heparin-Alexa Fluor 488 was purified on a PD-10 desalting column (GEHealthcare) equilibrated in autoclaved nanopure water. Fractionscontaining the labeled material detected at 490 nm were combined anddried overnight on a SpeedVac concentrator (Savant) with heating. Theresulting solid was dissolved in autoclaved nanopure water to 10 mg/mland stored at 4° C.

Heparin-Alexa Fluor 488 bound to PKH26- and DAPI-labeled C. albicans. Asingle colony of BWP17 wt was inoculated into 3 ml YPD medium andincubated overnight at 30° C. with shaking at 225 rpm. Using a PKH26 RedFluorescent Cell staining kit (including PKH26 dye and diluent C,Sigma), 2×10⁷ cells were mixed with 1 ml diluent C. To this solution wasadded a mixture of 4 μl PKH26 dye in 1 ml diluent C, and the mixtureallowed to stand at room temperature for 3 min. After adding 10 ml 3%BSA, the mixture was centrifuged at 3,000 rpm for 7 min. The resultantmagenta-colored cells were washed with 10 ml each of 1% BSA and PBS,with centrifugation after each step. After supernatant removal, thecells were reconstituted in 10 ml fresh PBS. Two 1 ml (2×10⁶ cells)aliquots were placed in separate 15 ml conical tubes, centrifuged, andsupernatants removed. Each was reconstituted with 2 ml YPD, andheparin-Alexa Fluor 488 (0.1 ml of above 10 mg/ml solution) was added tothe experimental sample. Control and experimental samples were incubatedat 30° C. with shaking (225 rpm) for 30 min, at which time 0.4 mlaliquots of each were removed, pelleted (10,000 rpm for 3 min), andwashed twice with PBS. After reconstitution with 0.5 ml PBS, DAPI(4′,6-diamidino-2-phenylindole dihydrochloride (Sigma, stock solution of5 mg/ml) was added to a final concentration of 1 μg/ml and the solutionallowed to stand at room temperature for 10 min. Cells were pelleted andwashed twice with PBS, then mounted on a microscope slide usingFluoromount G.

To measure the binding of C. albicans to solid-phase heparin, aheparin-binding ELISA assay was developed, as follows. A single colonyof each C. albicans wild type or mutant strain from a YPD plate wasinoculated into 3 ml YPD medium and incubated overnight at 30° C. withshaking at 225 rpm. Sigma heparin (fresh solution made daily), dilutedto 25 units/ml in autoclaved, sterile-filtered PBS was added as 0.1 mlaliquots to each well of an allyl amine-coated 96-well heparin bindingmicrotiter plate (BD Biosciences). The plate was incubated at roomtemperature overnight in the dark (50). In the morning, the plate waswashed with acetate buffer (100 mM NaCl, 50 μM NaOAc, 0.2% Tween 20, pH7.2), incubated with 3% bovine serum albumin (BSA) in PBS at 30° C. for1 hour, then washed with PBS. In the meantime, overnight cultures of C.albicans were subcultured by dilution to OD₆₀₀ of 0.2 in 25 ml of YPD,and grown at 30° C. to mid-log phase (OD₆₀₀ 0.6-0.7). Cells werepelleted (3,000 rpm for 7 minutes), washed twice with PBS andreconstituted in RPMI-HEPES to 4×10⁵, 2×10⁵, and 1×10⁵CFU/ml,respectively. One hundred μl of each C. albicans dilution was applied tothe microtiter wells; experiments for each strain were performed inquadruplicate. The plate was incubated at 30° C. for one hour and washedwith PBS to remove non-adherent C. albicans. One-tenth ml of abiotinylated polyclonal rabbit anti-C. albicans antibody raised againstsoluble proteins in a C. albicans lysate (Meridian Life Science), whichhad been diluted 1:2500 in FACS-Tween (0.05% Tween 20 in FACS bufferconsisting of 0.3% BSA), was added to each well. The plate was incubatedat 30° C. for one hour, then washed with PBS-Tween (0.05% Tween 20 inPBS). One-tenth ml streptavidin alkaline phosphatase (Biolegend) diluted1:10,000 in FACS-Tween was then added to each well, and the plate wasincubated at 30° C. for 30 minutes. After washing with PBS-Tween and AKPbuffer (100 mM Tris base, 50 μM MgCl₂, 100 mM NaCl, pH 9.5.), 0.1 mlalkaline phosphatase substrate (KPL) was added to each well. After 45minutes, absorbance at 595 nM was read on a Beckman Coulter DTX 880.When desulfated heparin analogs, chondroitin sulfate, or dermatansulfate were used, equimolar amounts of the analogs (with respect toheparin) were applied to the allyl amine-coated 96-well plate instead ofheparin.

The ability of soluble heparin to inhibit C. albicans binding toplate-bound heparin was demonstrated by pre-incubating 1×10⁶CFSE-labeled C. albicans with 200 units heparin at 37° C. for one hourin wells of a 96-well black plate. C. albicans was labeled withcarboxyfluorescein succinimidyl ester (CFSE), succinimidyl ester (CFSE;Invitrogen), which is used to track cell division and therefore does notkill the organism (28), as follows. A single colony of C. albicans wassuspended in CSM to an OD₆₀₀ of 0.1, diluted 1:200 into 25 ml CSM andgrown at 30° C. overnight to mid-log phase (OD₆₀₀ 0.6-0.7). Cells werewashed twice in sterile PBS and suspended in PBS at 2×10⁷ CFU/ml.Carboxyfluorescein succinimidyl ester (Life Technologies) was dissolvedin DMSO, diluted in PBS to 50 μM, and 0.5 ml CFSE mixed with 0.5 ml C.albicans suspension. The mixture was incubated for 20 min at roomtemperature on a rotator, washed once with FACS buffer, once with PBS,then suspended in 1 ml FACS buffer. Intensity of labeling was determinedas the mean fluorescence intensity using a BD Accuri C6 cytometer (SanJose, Calif.) with excitation at 488 nm and a 533/30 emission filter.Wells of a black 96-well microtiter plate were incubated with 0.1 mgpoly-D-lysine for one hour at room temperature, washed three times withPBS, then incubated with 200 units of heparin in 100 μl RPMI-HEPESovernight at room temperature. The following afternoon, C. albicans wildtype strain and mutants were labeled with CFSE as follows: after growthof C. albicans to mid-log phase in CSM medium, organisms were washed twotimes in PBS and suspended in PBS at a concentration of 2×10⁷/ml. 0.5 mlcells was mixed with 0.5 ml CFSE, covered in foil, and rotated on amixer for 20 minutes at room temperature. CFSE-labeled C. albicans cellswere pelleted in a minifuge at 3000 rpm for four minutes, washed oncewith FACS buffer (0.3% bovine serum albumin in PBS), washed again withPBS, and then suspended in 1 ml FACS buffer at a concentration of1×10⁷/ml. 100 μl CFSE-labeled C. albicans (1×10⁶) were deposited in testwells for 60 minutes at room temperature. At the end of the incubationperiod, wells were washed three times with PBS, and fluorescence in eachwell was measured (Beckman Coulter Multimode detector DTX880).

FIGS. 3A-D demonstrate C. albicans binding to heparin in vitro. FIG. 3Ashows confocal microscopy of C. albicans in the presence ofheparin-Alexa Fluor 488, with (i) C. albicans cell wall stained withPKH26, (ii) nucleus stained with DAPI, (iii) cell wall outlined byheparin-Alexa Fluor 488, and (iv) arrows indicating co-localization ofheparin with C. albicans cell surface. Incubation of C. albicans with100 units/ml heparin-Alexa Fluor 488 for 30 mins at 30° C. demonstratedco-localization of heparin with the C. albicans cellular surface (FIG.3A). Heparin deposition was seen at the interface of adjoining yeastcells and in individual cells.

FIG. 3B shows binding of 10,000 colony-forming units (CFU) C. albicans(OD₅₉₅) to increasing concentrations of heparin immobilized on an allylamine-coated 96-well microtiter plate; values are ±SD of duplicatewells. Binding of C. albicans at 10,000 CFU/well did not change overheparin concentrations ranging from 1.25 units/well (6.3 μg/well) to 20units/well (100 μg/well) (FIG. 3B), indicating saturation of the allylamine plate by heparin in low concentrations.

FIG. 3C shows the linear dose-response for the heparin binding ELISAassay with 2.5 units/well heparin and increasing C. albicans input from10,000 (left-most bar), 20,000 (center bar), and 40,000 (right-most bar)CFUs; the graph represents mean±SD of four experiments, *p<0.007 for allinputs. FIG. 3C shows that the OD₅₉₅ of the ELISA assay increaseslinearly as input of C. albicans increased from 10,000 colony formingunits (CFU)/well to 40,000 CFU/well.

FIG. 3D shows binding of C. albicans (40,000 CFU/well) to equimolaramounts of heparin analogs desulfated at the 2-O (second from the leftbar), 2-N (second from the right bar), or 6-O (right-most bar) positionsversus heparin control (left-most bar) (normalized to 100%), with thegraph representing mean±SD of three experiments, performed in triplicate*p≦0.003, ** p=0.20 vs. heparin control. With equimolar amounts ofdesulfated heparins, binding of C. albicans was decreased by 11% whenheparin was desulfated at the 2-0 position of iduronic acid (p=0.003),and by 21% when heparin was desulfated at the 2-N position ofglucosamine (p=0.002) (FIG. 3D). Removal of the sulfate group at the 6-Oposition of glucosamine did not significantly reduce binding.

Pre-incubation of CFSE-labeled C. albicans with 100 units/ml heparindecreased binding to heparin by 27% (p=0.022; data not shown). C.albicans also recognizes the related structures of chondroitin sulfateand dermatan sulfate. Binding of C. albicans to heparin and tochondroitin sulfate was equivalent, but binding to dermatan sulfate wasreduced (p=0.019; data not shown). These results confirmed that thebinding of C. albicans to heparin and similar glycosaminoglycans(chondroitin sulfate and dermatan sulfate) could be reproduciblymeasured and, in the case of heparin, specifically inhibited bypre-incubating the organisms with heparin. These results also suggestedthat surface components of C. albicans may mediate C. albican binding toheparin.

Location of HBM in Int1 (SEQ ID NO: 5) were determined. Five overlappingpolypeptides spanning amino acids 51-1711 of Int1 were expressed with a6×His tag (SEQ ID NO: 54) in S. cerevisiae BJ3501 and purified byaffinity chromatography (HisTrap column; GELifesciences). Fractionscontaining the His-tagged polypeptide were pooled, diluted with loadingbuffer (10 mM phosphate, pH 7.0 plus 250 mM NaCl) and applied to aheparin sepharose column (HiTrap Heparin HP; GE Lifesciences).Heparin-binding polypeptides were eluted with a step gradient of NaCl(0.5-2 M) in loading buffer. Eluted fractions were analyzed by SDS-PAGEand immunoblot using anti-His tagged antibody (Santa Cruz) andchemiluminescent detection (SuperSignal West Pico Mouse IgG DetectionKit, Pierce) according to manufacturers' instructions. Polypeptidesspanning aa 656-1193 and aa 1548-1711 bound to heparin-Sepharose andwere eluted with NaCl, indicating that the HBM in those domains,schematized as Motif 1 (SEQ ID NO: 1), Motif 4 (SEQ ID NO: 2), and Motif5 (SEQ ID NO: 3) (FIG. 4A), were candidates for mediating binding toheparin. Bolded letters represent basic amino acids in heparin bindingmotifs; unbolded letters represent hydropathic residues. Polypeptidesspanning aa 51-385, aa 385-659, and 1188-1551 failed to bind to aheparin-Sepharose column.

The linear polypeptide spanning aa 656-1193 contains one potentialheparin binding site, ₈₀₄QKKHQIHK (SEQ ID NO: 1) (basic residues shownin red) (Motif 1 in Table 1 and FIG. 4A). The linear polypeptidespanning aa 1548-1711 contains a canonical Weintraub motif ₁₅₉₃FKKRFFKL(SEQ ID NO: 2) (Motif 4 in Table 1 and FIG. 4A) and a canonical Cardinmotif ₁₆₁₂SHKTRA (SEQ ID NO: 3) (Motif 5 in Table 1 and FIG. 4A).Sequence homology search using BLAST indicated that aa 1548-1711contains a Pleckstrin homology domain (PHD), which is structurallyresolved. Using homology-based 3-D modeling, we found that the threelysine residues and single arginine residue in Motif 4 were located onthe rim of the PHD and might facilitate binding to a strong anion suchas heparin by electrostatic interaction (FIG. 4B, arrows). The Cardinmotif (Motif 5) did not share this conformation.

To test whether ₈₀₄QKKHQIHK (SEQ ID NO: 1) (Motif 1) and/or ₁₅₉₃FKKRFFKL(SEQ ID NO: 2) (Motif 4) mediated the binding of heparin to C. albicans,standard PCR-mediated mutagenesis (51) was used to construct a set ofisogenic INT1 disruptants and mutants (Table 2).

TABLE 2 Strain Abbreviation Genotype Source BWP17 —ura3::imm434/ura3::imm434 his1::hisG/his1::hisG [27]arg4::hisG/arg4::hisG BWP17WT WT BWP17 plusarg4::ARG4::URA3/his1::hisG::HIS1 [27] VBIDM2 — as BWP17 plusint1::ARG4/int1::URA3 [60] VBIDM6-2 DD as VBIDM2 plus his1::hisG/HIS1[60] KO503 Motif 4 VBIDM2 plus his1::hisG::HIS1-INT1 (KK1595AA) currentKO507 Motif 1 VBIDM2 plus his1::hisG::HIS1-INT1(KK804AA) current KO508Motifs 1&4 VBIDM2 plus his1::hisG::HIS1-INT1(KK804AA, KK1595AA) currentKO509 Reint VBIDM2 plus his1::hisG::HIS1-INT1 (WT) current

Construction of mutants was as follows. C. albicans genomic DNA wasisolated from saturated overnight cultures using glass beads asdescribed (52). A lithium acetate method was used to transform C.albicans (41). Plasmids and PCR products were purified using kits(Fermentas/ThermoFisher, Pittsburg Pa.) or established methods (53). Pfuenzyme (New England Biolabs) with High Fidelity buffer was employed forall amplifications. Products were sequenced to affirm fidelity prior touse. Primers are described in Table 3.

TABLE 3  Primer Internal reference Sequence Purpose 1 SAC2035UPgggagctcGTTACTTGTCATTAATTAGTTACTTCC SacI 5′ INTI (SEQ ID NO: 55) 2MLU3′UTR ggacgcgtTTTTATCTTTTTATGTAAATATATACTA MluI 3′INT1(SEQ ID NO: 56) 3 INT1 KR1595AA F ATTGTCCAATTTTTAAGGCTGCTTTTTTCAAATTAATmutate Motif 1 GGG (SEQ ID NO: 57) 4 INT1 KR1595AA RCCATTAATTTGAAAAAAGCAGCCTTAAAAATTGGAC mutate Motif 1 AATC (SEQ ID NO: 58)5 INT1 KK805AA F GCATAAACAAGAAAAGCAGGCCGCCCATCAAATTC mutate Motif 4ATAAAGTTCC (SEQ ID NO: 59) 6 INT1 KK805AA RGGAACTTTATGAATTTGATGGGCGGCCTGCTTTTC mutate Motif 4TTGTTTATGC (SEQ ID NO: 60) 7 GHISR CTCCCGGCCGCCATGGCCGC (SEQ ID NO: 61)check integration 8 HIS3AMP GTTGGTGTGGCCCAGAGACTCT (SEQ ID NO: 62) checkintegration

A single copy of Int1, including 1450 bp upstream and 548 bp downstreamfrom the Int1 open reading frame (www.candidagenome.org, Assembly 21),was integrated into the hisG locus of the int1−/− strain VBIDM2 (54) toproduce the reconstituted strain KO509. Briefly, a copy of Int1 wasgenerated by PCR, using primers 1 and 2 with BWP17 wt DNA as templateand cloned into the SacI/MluI sites of pGEMHIS (51) to create pKO509.pKO509 was digested with SwaI and transformed into VBIDM2 to create thereconstituted strain KO509. PCR-mediated overlap extension mutagenesis(55) was used to produce copies of Int1 mutated at putative heparinbinding domains. Briefly, primer pairs 1+3 and 2+4 (or 1+5 and 2+6) wereused to produce two overlapping fragments of INT1 in which putativeheparin binding domains were mutated (FKKRFFKL (SEQ ID NO: 2)→FKAAFFKL(SEQ ID NO: 53) or KQKKHQ (SEQ ID NO: 64)→KQAAHQ (SEQ ID NO: 63)), and afull length mutated sequence generated in a third per using primers 1+2with the fragments as template. The mutated sequences were cloned intothe SacI/MluI sites of pGEMHIS to create pKO503 and pKO507,respectively. A construct mutated at both sites (pKO508) was producedusing primers 1+5 and 2+6 with plasmid DNA from pKO503 as template. Fulllength mutated products cloned into pGEMHIS and used to transform VBIDM2as above, producing strains KO503, KO507, and KO508. The correctinsertion and orientation of all constructs was confirmed by PCR. Ingrowth curves performed with and without 100 units/ml heparin at 30° C.in RPMI-HEPES, there was no difference in the doubling times of the wildtype, Δint1 mutant, INT1 reintegrant, or the reintegrants containingalanine substitutions in Motif 1 (₈₀₄QKKHQIHK (SEQ ID NO: 1) to QAAHQIHK(SEQ ID NO: 52)), Motif 4 (₁₅₉₃FKKRFFKL (SEQ ID NO: 2) to FKAAFFKL (SEQID NO: 53)), or Motifs 1 and 4. Percent binding of each mutant toheparin in the ELISA assay was compared to the wild type according tothe formula [Absorbance₅₉₅ mutant]/[Absorbance₅₉₅ wild type]×100.

The ELISA assay used a commercially available 96-well microtiter platecoated with polymerized allylamine (BD Biosciences) (27), in which wellswere inoculated with 2.5 units (12.5 μg) heparin in 100 μl phosphatebuffered saline (PBS) per well. The plate was covered and allowed tostand at room temperature overnight, then washed with acetate buffer(100 mM NaCl, 50 mM NaOAc, 0.2% Tween, pH 7.2). Wells were washed threetimes with PBS, then inoculated with 1×10⁴ to 4×10⁴ Candida albicansyeast cells that were grown overnight at 30° C. in yeast peptonedextrose (YPD) broth, washed, and resuspended in RPMI-HEPES at 1×10⁷organisms/ml. The plate was then incubated at 30° C. for one hour, thenwashed with PBS-Tween (PBST, PBS/0.05% Tween). A commercially availablebiotinylated anti-Candida antibody (Meridian Life Science, MemphisTenn.) was diluted 1:1500 in FACS-TWEEN (0.3% BSA/0.05% Tween), added tothe wells, incubated at 30° C. for one hour, then washed with PBST.Streptavidin-alkaline phosphatase (BioLegend, San Diego Calif.) was thenadded to the wells at a 1:10,000 dilution in FACS-TWEEN and incubated at30° C. for 30 min, then washed with PBST and AKP buffer (100 mM Tris,100 mM NaCl, 50 mM MgCl₂*6H₂O). BluePhos^(@) microwell substrate(Kirkegaard and Perry Laboratories, Gaithersburg Md.) was added to thewells and allowed to react for 45 min at room temperature. The colorchange was read spectrophotometrically at absorbance of 595 nm.

FIGS. 4A-D show that linear heparin binding motifs in C. albicans Int1mediate binding to heparin.

FIG. 4A illustrates the five putative heparin binding sites in Int1.Mutations were made by substituting alanine residues in Motif 1:₈₀₄QKKHQIHK (SEQ ID NO: 1) to QAAHQIHK (SEQ ID NO: 52) (Table 1,Motif 1) and in Motif 4: ₁₅₉₃FKKRFFKL (SEQ ID NO: 2) to FKAAFFKL (SEQ IDNO: 53) (Table 1, Motif 4). A third reintegrant had alaninesubstitutions at both sites.

FIG. 4B shows molecular modeling of Motif 4, which predicts that thethree lysine residues and the single arginine residue (arrows) mightfacilitate binding to a strong anion like heparin.

FIG. 4C shows heparin binding of C. albicans WT (normalized to 100%),Δint1 double disruptant (DD), and four single-copy reintegrants: (1)Reint contains one wild type copy of Int1; (2) Motif 1 mutant containsalanine substitutions for lysine residues at positions 805/806(₈₀₄QAAHQIHK (SEQ ID NO: 52)); (3) Motif 4 mutant contains alaninesubstitutions for lysine 1595 and arginine 1596 (₁₅₉₃FKAAFFKL (SEQ IDNO: 53)), and (4) Motif 1&4 mutant contains alanine substitutions atboth sites. Binding to heparin was highest for the wild-type C. albicansstrain that expressed both copies of the INT1 gene. Disruption of bothcopies of INT1 (DD) reduced the binding of heparin by 40% (p=0.031)despite removing just one of the 34 C. albicans genes encoding putativelinear HBM. Reintegration of one copy of the wild type INT1 gene (Reint)partially restored the ability of C. albicans to bind to heparin.Differences in heparin binding among the WT, Reint and DD strains werestatistically significant (p=0.031 in all cases). Among the threeisogenic strains with alanine mutations in putative heparin bindingsites, mutation of Motif 1 was associated with the largest reduction inheparin binding, compared to the wild type strain (p=0.031), to theMotif 4 mutant (p=0.036), and to the Motif 1&4 mutant (p=0.031). Resultswith the Motif 1 mutant were not significantly different from thepercent binding observed with the double disruptant (p=0.063) or thereintegrant (p=0.115), nevertheless, this trend of reduced bindingsuggested that lysine residues in Motif 1 might be likely mediators ofheparin binding. Results are presented as mean±SD for n=5 experimentsperformed in quadruplicate, * p=0.031, ** p=0.036.

Because Motif 1 in Int1 appeared to mediate a considerable proportion ofthe binding of C. albicans to heparin, a peptide encompassing this motifHKQEKQKKHQIHKV (SEQ ID NO: 4) was used to immunize rabbits with acommercial protocol (Pacific Immunology). Affinity-purified immune IgGand IgG from pre-immune rabbit serum (both from Pacific Immunology) werecompared for the ability to inhibit binding of C. albicans to heparin(FIG. 5). Antibody and heparin inhibition studies were performed asfollows. Antibody was produced and tested by Pacific Immunology, Ramona,Calif. (www.pacificimmunology.com; NIH Animal Welfare Assurance NumberA41820-01; USDA License 93-R-283). A peptide corresponding to aminoacids 799-812 of Int1 (HKQEKQKKHQIHKV (SEQ ID NO: 4)), encompassingMotif 1, was conjugated to KLH via an N-terminal cysteine residue andused to immunize two NZW rabbits. The animals were immunized once withconjugated peptide in a proprietary formulation of Freund's completeadjuvant and boosted 3 times with conjugated peptide in Freund'sincomplete adjuvant. The same peptide was conjugated to CNBr-Sepharoseand used for affinity purification of epitope-specific IgG. The finalserum titer for both animals was >1:100,000 by ELISA. Both pre-immunerabbit serum and serum from bleed 3 were chromatographed on a 2 mlProtein A column (Thermo Scientific) to yield pre- and post-immune IgGand brought to a concentration of 1.6 mg/ml. 100 μl poly-D-lysine (MPBiomedicals, Solon Ohio) diluted as 1 mg in 10 ml distilled water wasadded to each well of black 96-well plates (Costar) and incubated atroom temperature for 60 min. After three washes with PBS, 100 μlpharmacologic heparin (2000 units/ml in RPMI-HEPES) was added to eachwell and incubated overnight at room temperature in the dark. In themorning, the plate was washed three times with PBS, then a 1:1000dilution of pre- or post-immune IgG in PBS was added to each well andincubated for 30 min at room temperature. After overnight growth tomid-exponential phase in CSM, C. albicans wild type and doubledisruptant strains were labeled with CFSE as above. After labeling,samples of each strain were removed to determine the intensity oflabeling by flow cytometry, as described above. CFSE-labeled C. albicanswere diluted in RPMI-HEPES to yield 1×10⁶ cells per 100 μl and added toeach well for 60 min at 30° C. The plate was washed three times withPBS; fluorescence in each well was measured with a Beckman DTS 880.Experiments were performed in triplicate. For heparin inhibitionstudies, wild type C. albicans were grown as above, labeled with CFSE,and washed to remove excess CFSE. Organisms were pelleted, thesupernatant was removed, and the pellets were suspended in 2 mlpharmacologic heparin (20,000 units/ml) diluted with RPMI-HEPES to aconcentration of 250 units/ml (1.3 mg/ml). CFSE-labeled C. albicans wereincubated with heparin for 60 min at 37° C. with shaking at 400 RPM,then added to the wells of a black plate, incubated for 60 min at RT,and washed three times with PBS; fluorescence in each well was measuredwith a Beckman DTS 880. The assay was performed in a 96-well black platewith CFSE-labeled C. albicans. Wells were coated with poly-D-lysine, 100μg/100 μl PBS for 60 min at room temperature, then 200 units heparin in50 μl RPMI-HEPES were added to each well. The plate was incubatedovernight at room temperature. The following afternoon, pre-immune IgGand post-immune IgG were diluted in PBS to a final concentration of 13.2μg/ml. 50 μl of pre- or post-immune IgG was deposited in the appropriatewell for 30 min at room temperature, then 1×10⁶ CFSE-labeled C. albicansin 50 μl RPMI-HEPES were added for two hours at room temperature. Afterthis incubation, the wells were washed three times with PBS, andfluorescence was measured on the multimode detector as previouslydescribed.

Polyclonal IgG to the HKQEKQKKHQIHKV (SEQ ID NO: 4) motif in domain 3 ofInt1 blocked 19% of binding of wild type C. albicans (WT) to heparin.FIG. 5 shows heparin binding of wild type C. albicans preincubated withpre-immune IgG (left closed circles) and wild type C. albicanspre-incubated with post-immune IgG raised to the polypeptideHKQEKQKKHQIHKV (SEQ ID NO: 4) (right closed circles), where datarepresent mean±SD of n=15 experiments performed in duplicate, * p=0.023,by paired statistics. These results confirmed the importance of Motif 1,as a mediator of heparin binding in C. albicans.

In other systems, heparin has been reported to cleave surface proteins(56), change protein conformation (57), and bind trypsin-sensitivelysine and arginine residues in histone H1 (58). In order to understandwhether incubation with heparin changed the conformation of C. albicanssurface proteins, 1×10⁷ C. albicans were incubated for one hour at 3T Con a rotator with pharmacologic heparin (20,000 units/ml) diluted inRPMI-HEPES to a working concentration of 250 units/ml. C. albicans 1×10⁷C. albicans in an equal volume of RPMI-HEPES served as control.Supernatants were removed. 300 μl of Hep+ and Hep− supernatants wereincubated with 100 μl avidin agarose beads (Thermo Scientific) for 60min at room temperature on a rotator in the presence of 50 units heparin(Hep+); an equal volume of RPMI-HEPES was substituted for heparin in theHep− supernatants. Beads were pelleted, and 100 μl beads were incubatedwith 100 μl 3.0 M NaCl for 30 mins at room temperature on a rotator.Beads were pelleted and the supernatants were withdrawn; 100 μl TCA wasadded to 100 μl Hep+ or 100 μl Hep− supernatants and incubated on iceovernight at 4° C. The TCA precipitates were stored at −80° C. untilanalysis by mass spectroscopy.

Six biological replicates of TCA precipitated protein pellets from equalcellular equivalence of heparin-treated (Hep+) and untreated (Hep−)conditioned medium from cultures of C. albicans were each solubilized in50 μL Laemmli gel buffer. To remove any residual TCA and to furtherconcentrate the samples, each was subjected to buffer exchange andconcentration using an Amicon ultra 3 kDa microfuge filtration cartridgeat 14,000×g for 15 min with five subsequent additions of 50 μL of 1×Laemmli gel buffer between spins. The resulting retained proteins (6Hep+ and 6 Hep−) were subsequently prepared for SDS-PAGE by combininginto two pools of three samples for the Hep+ and Hep− conditionedmedium. The replicate sample pools were loaded onto two 4-12% mini gelsand separated using the MOPS buffer system followed by silver stainingto visualize the proteins using the Sigma Proteosilver system. Theproteins were prepared for identification and quantitation by massspectrometry by gridding the gel lanes from the replicates of Hep+ andHep− samples into 11 equal regions followed by in gel trypsin digestionand extraction of peptides as described (59)

The recovered peptides from the gridded gel sections were analyzed byliquid chromatography coupled nano-electrospray mass spectrometry(nLC-MSMS) on a TripleTOF 5600 mass spectrometer (AB Sciex, TorontoCanada) attached to an Eksigent (Dublin Calif.) nanoLC ultra nanoflowsystem. Recovered peptides from each fraction were loaded on toIntegraFrit Trap Column (outer diameter of 360 μm, inner diameter of100, and 25 μm packed bed) from New Objective, Inc. (Woburn Mass.) at 2μl/min in FA/H2O 0.4/99.2 (v/v) for 10 min to desalt and concentrate thesamples. For the chromatographic separation of peptides, the trap-columnwas switched to align with the analytical column, Acclaim PepMap100(inner diameter of 75 μm, length of 15 cm, C18 particle sizes of 3 μmand pore sizes of 100 Å) from Dionex-Thermo Fisher Scientific (SunnyvaleCalif.). The peptides were eluted using a linear gradient from 95% phaseA (FA/H2O 0.4/99.6, v/v) to 40% phase B (FA/ACN 0.4/99.6, v/v) from 5min to 22.5 min (2% ACN/min) at a flow of 300 mL/min. As the peptideseluted from the column they were sprayed into the mass spectrometerusing NANOSpray® III Source (AB Sciex, Toronto Canada). Ion source gas 1(GS1), ion source gas 2 (GS2) and curtain gas (CUR) were respectivelykept at 7, 0 and 25 vendor specified arbitrary units. Interface heatertemperature and ion spray voltage was kept at 150 C, and at 2.3 kVrespectively. Mass spectrometer method was operated in positive ion modeset to go through 25 minutes, where each cycle performing one TOF-MSscan type (0.25 sec accumulation time, in a 400 to 1600 m/z window)followed by twenty information dependent acquisition (IDA)-modeMS/MS-scans on the most intense candidate ions selected from initiallyperformed TOF-MS scan during each cycle having a minimum 150 counts.Each product ion scan was operated under vender specifiedhigh-sensitivity mode with an accumulation time of 0.05 sec and a masstolerance of 50 mDa. Former MS/MS-analyzed candidate ions were excludedfor 10 sec after its first occurrence, and data were recorded usingAnalyst®-TF (1.5.1) software.

NanoLC-MSMS data collected from the Hep+ and Hep− samples were convertedto Mascot generic files and searched against the SwissProt fungaldatabase on an in house server running Mascot version 2.2.07 (MatrixScience, Ltd). Specific search parameters included up to two missedtryptic cleavages, carbamidomethylation of Cys, oxidation of Met,peptide and fragmentation mass tolerance of 0.1 Da. Only proteins with aminimum of two peptides with Mascot peptide score indicating a peptideidentity and a false discovery rate (FDR) against and inverse databaseat less than 1% were reported. Semi-quantitative measurements betweenthe Hep+ and Hep− proteins were generated using a minimum of two trypticpeptides from each protein as surrogates for the amount of proteins fromthe two groups. This was accomplished by capturing extracted ionprofiles for each peptide and then comparing the mono-isotopic peakintensity at the apex of the signal for the M+2H or M+3H signal for eachpeptide. An average ratio of the maximal peak intensity between twoindependent peptides for each protein presented was used to determinesemi-quantitative ratios of proteins between the Hep+ and Hep− samples.

To determine the levels of these proteins in control versusheparin-treated organisms, the relative level of several trypticpeptides were used as surrogates for the protein levels.

Compared to untreated organisms, heparin treatment led to a seven-foldincrease in intensity of IDVVDQAK (SEQ ID NO: 65) from Eno1 (FIG. 6A), aten-fold increase in intensity of SLLDAAVK (SEQ ID NO: 66) from Pgk1(FIG. 6B), and a five-fold increase in VPTTDVSVVDLTVR (SEQ ID NO: 67)from Tdh3 (FIG. 6C) in supernatants.

Other peptides from Eno1, Pgk1, and Tdh3 also exhibited increasedintensities, ranging from 10-16 fold the intensity of the correspondingpeptides from untreated organisms (data not shown), further validatingsignificant increases in the protein levels in the supernatants forheparin-treated cultures. Of the 12 proteins whose peptides were foundin highest concentration in the supernatant (Table 4), all but one areknown to be localized to the cell wall; cellular localization of Eft1 isnot known. Eight of the twelve, including Eno1, Pgk1, Tdh3 and Ssa1/2,themselves contain putative heparin binding motifs (Table 1) and areconsidered critical antigens for innate and adaptive immune responsesagainst C. albicans (31-36).

TABLE 4 ID Prot Score Prot Mass Peptide Matches** Eno1*^(,#) 2877 47202213 Ssa2*^(,#) 1938 70199 73 Pgk1*^(,#) 1826 45266 131 Tdh3*^(,#) 175135508 192 Hsp90^(#) 1666 80773 65 Ssb1*^(,#) 1608 66562 82 Met6^(#) 156685763 70 Ssa1*^(,#) 1455 70452 61 Adh1^(#) 1218 37255 67 Eft1 1187 5042699 Ino1*^(,#) 1006 57857 39 Eft2*^(,#) 933 93865 25 *proteins withputative linear heparin binding motifs; **total peptides identified byMSMS including duplicates; and ^(#)cell wall proteins by Gene Ontology.

In order to evaluate the consequences of removal of Ssa1 and Ssa2 (Table4), both targets of histatin, a histatin killing assay was performed asfollows. C. albicans strains were grown to late log phase overnight inYPD. A single colony was suspended in 1 ml YPD, diluted 1:500 into 10 mlYPD, and incubated overnight at 30° C. and 225 rpm to OD₆₀₀ 1.0. Yeastcells were washed twice in PBS and 2×10⁴ cells suspended in 250 μlRPMI+10 mM HEPES, pH 7.0, with or without 500 units/ml heparin (Sigma).The cells were incubated at 37° C. for one hour with shaking, washedtwice with 10 mM phosphate buffer, pH 7.4, and suspended in 20 μlphosphate buffer. Histatin 5 (final concentration 15 μM; PeptidesInternational) or 10 mM phosphate buffer was added to the preincubatedcells (total volume 40 μl) and incubated further at 37° C. for 90minutes with shaking. YPD (360 ul) was added to each tube, a 40 μlaliquot spread onto YPD plates, and colonies counted after two days. Theeffect of heparin treatment was determined by the formula %change=[(cfu+heparin)−(cfu−heparin)]/(cfu+heparin).

Heparin binding to trypsin-sensitive lysine and arginine residues inhistone H1 unfolds chromatin and increases its accessibility (58). Totest whether heparin binding could influence gene expression in C.albicans, qRT-PCR was performed on a selective set of 13 genes involvedin adhesion, cell-cell interaction, and biofilm formation afterincubating C. albicans with and without 100 units/ml heparin at 37° C.for 75 minutes. Gene expression studies were performed as follows.Overnight cultures (3 ml YPD at 30° C., 225 rpm) were diluted in 25 mlYPD to a nominal concentration of 8×10⁵/ml and grown to OD₆₀₀approximately 1.0 (30° C., 225 rpm), collected by centrifugation, andwashed in PBS. Cells (5×10⁷) were suspended in 5 ml RPMI1640 with 25 mMMOPS pH7.4, with or without 100 units/ml Sigma heparin in 50 mlpolypropylene tubes, and incubated at 37° C. with shaking for 75minutes. One ml aliquots (approximately 10⁷ cells) were collected bycentrifugation, washed once with room temperature PBS, and frozen at−80°. Pellets were thawed on ice and suspended in 1 ml Tri-Reagent (MRCResearch) in a 2 ml screw capped tube containing about 0.2 g acid washedglass beads (Sigma) and vortexed three times for 1 minute with a 1minute rest on ice between each vortex. The lysates were rested for fivemin at room temperature, centrifuged for five min at 12,000 rpm, and RNAisolated from the supernatant using the DirectZol kit (Zymo), includingDNAse digestion, per manufacturer's instructions. cDNA was produced fromequivalent quantities of RNA (between 300 and 900 ng) for each treatmentusing the Maxima Reverse Transcriptase Kit for qRT-PCR (Fermentas). Twoμl of 1:5 dilution of cDNA was used in each qPCR reaction with 500 nM ofeach primer and Fast SYBR Green Master Mix (Life Technologies) on a 7500FAST Instrument (Applied Biosystems) according to manufacturer'sinstructions. Relative expression was determined using the ΔΔCt method(60) with 18S RNA as the reference. Primers are shown in Table 5.

TABLE 5  Primer Sequence SEQ ID NO: 18S F TCTTGTGAAACTCCGTCGTG 68 18S RAGGGACGTAATCAACGCAAG 69 AHP1 F TGTGCCTGGTGCTTTTACC 70 AHP1 RTTAGCCCAAGCTGCCATTAC 71 ALS1 F TCATTTGCCACCACTACCAC 72 ALS1 RTGGCATAGGATTGTGACCAG 73 ALS3b F GCTGGTGGTTATTGGCAACGTGC 74 ALS3b RTGGTAAGGTGGTCACGGCGG 75 CDC10 F AGATCAAGGGCAAACCTCAC 76 CDC10 RATAGGAGCATTTGGCACACC 77 EAP1 F TACCCAGGCCAATACAAAGG 78 EAP1 RTAATGGGCTTGACCTTGGAG 79 ECE1 F CTAATGCCGTCGTCAGATTG 80 ECE1 RAACATCTGGAACGCCATCTC 81 ENO1 F CCATTGACAAAGCCGGTTAC 82 ENO1 RTTAGATGGGTCGGATTCTGG 83 HGC1 F AGGTCGCAAGCAACAACAAC 84 HGC1 RAAGAAACAGCACGAGAACCAG 85 HWP1b F TCCTGCCACTGAACCTTCCCCAG 86 HWP1b RCCACTTGAGCCAGCTGGAGCG 87 HWP2 F CCACCAAAACCAAGTGCTAC 88 HWP2 RAACTCCAGATGATCCCGAAG 89 INT1 F TGTGCCCACTGAAGTCAAAG 90 INT1 RGCTTTACCGGTGATTTGGTC 91 RBT1 F CACCTCATGCTCCAACAATG 92 RBT1 RGATGATTCTGGGGCTGATTC 93 RBT5 F TGCTGAAAGTTCTGCACCAG 94 RBT5 RGCTTCAACGGAAACAGAAGC 95

FIG. 7A shows CFU of C. albicans WT and DD after 75-minute incubationwithout (left bar of each set) or with (right bar of each set) 500units/ml heparin followed by histatin 5 (15 μM), performed in duplicate.Confirming proteomic results showing removal of Ssa1/2 (Table 3),incubation of wild type C. albicans with 500 units/ml heparin led to a25% decrease in histatin-mediated killing (FIG. 7A); in contrast,heparin treatment of the INT1 double disruptant did not impair killing(1.5% decrease).

FIG. 7B shows relative mRNA expression of thirteen genes measured byqRT-PCR after incubation of C. albicans with heparin, with the level ofexpression compared to C. albicans without heparin (=1). Results showeda 2.5 to 3-fold increase in mRNA for HWP1 and HWP2.

In order to understand whether heparin binding motifs 1 and 4 influencedC. albicans pathogenesis in vivo, we employed a rat model of biofilmformation in central venous catheters inserted into the jugular vein, aspreviously described (61). After insertion into the jugular vein of ananesthetized female Sprague-Dawley rat, a silastic catheter washeparinized with 100 units heparin/mil and remained in place for 24hours. At the 24 hour timepoint, 500 μl of blood was withdrawn andcultures to insure sterility, and then 1×10⁶ CFU of the desired C.albicans strain was instilled into the catheter and allowed to dwell for6 hours. The animal was then sacrificed, the catheter was removedaseptically and processed for scanning electron microscopy. Biofilmformation on the intra-luminal surface of the catheter was assessed byscanning electron microscopy (SEM) at 100× and 2000×.

FIGS. 8A-F demonstrated that heparin binding motifs contribute tobiofilm formation in vivo.

The INT1 wild type strain (WT) produced a profuse biofilm withintertwined hyphae and visible exopolysaccharide matrix (FIG. 8A).Biofilm formation by the Int1 double disruptant was much reduced on SEM(FIG. 8B), as expected. Reintegration of one wild type copy of INT1restored a profuse biofilm (FIG. 8C). However, alanine substitution oflysines_(805/806) in Motif 1 greatly impaired biofilm formation (FIG.8D). Although alanine substitution of lysine₁₅₉₅ and arginine₁₅₉₆ inMotif 4 did not reduce biofilm formation (FIG. 8E), the Motif 1&4 mutantagain produced sparse biofilm (FIG. 8F). These results showed thatlysine residues 805/806 in Motif 1 were critical for biofilm formationin vivo.

Inhibition of biofilm formation by the antibody raised against thepeptide sequence HKQEKQKKHQIHKV (SEQ ID NO:4) was also tested in the ratcentral venous catheter model. A 1:10 dilution of affinity-purified IgGagainst the peptide HKQEKQKKHQIHKV (SEQ ID NO: 4) was incubated withwild type C. albicans at 30° C. for one hour. A 1:10 dilution ofpre-immune IgG was used as a control. The strains were then instilledinto separate jugular venous catheters in individual rats. After 6hours, catheters were removed and aseptically processed for scanningelectron microscopy (100× and 2000×) as described above.

Central venous catheters from animals that received C. albicanspre-incubated with IgG against HKQEKQKKHQIHKV (SEQ ID NO:4) exhibitedsubstantially reduced biofilm formation. FIG. 9 (left panel) showsintraluminal biofilm (100× and 2000×) from C. albicans incubated withpre-immune IgG; there is no diminution in biofilm, hyphae, or productionof exopolysaccharide matrix. FIG. 9 (right panel) shows intraluminalbiofilm (100× and 2000×) from C. albicans incubated with post-immune IgGrecognizing the sequence HKQEKQKKHQIHKV (SEQ ID NO: 4); there is amarked diminution in biofilm with sparse hyphae and no matrix.

In vitro study results showed that C. albicans binds heparin through HBMin Int1 (FIG. 4C). The specificity of this interaction was confirmed byinhibition with heparin (data not shown) and with antibodies directedagainst a peptide that encompasses Motif 1 in Int1 (FIG. 5). Binding ofheparin results in several consequences that could potentially impactvirulence in vivo: removal of Candida surface proteins that serve astargets for innate (FIG. 7A) and adaptive (FIG. 6A-C) immune defensesand modulation of gene expression (FIG. 7B).

The in vivo studies of biofilm formation in heparinized central venouscatheters in rats showed an obvious reduction in biofilm formation aftermutation of lysine residues 805/806 in Motif 1 (FIGS. 8D, 8F). Inaddition, a rabbit IgG antibody directed against a peptide encompassingMotif 1 dramatically inhibited biofilm formation in the rat centralvenous catheter model as well (FIG. 9, right panel). These results notonly demonstrate the central role of lysine residues in Motif 1 but alsohave important clinical implications because of the use of heparin incentral venous catheters, in which setting Candida spp. are the fourthmost common cause of infections (23, 24).

Putative linear HBM are also present in Staphylococcus epidermidis andStaphylococcus aureus (Long and Hostetter unpublished data), twoorganisms that are even more common causes of catheter-associatedinfection (43), as shown in Tables I-IV. For example, putative linearHBM were identified in the following cell wall or putative cell wallproteins, where the motif and the beginning position of the motif isindicated in parenthesis, from methicillin resistant Staphylococcusaureus, strain 252 (MRSA252): sasC (LKKNKY; 4; and IRKYKV; 11), isdB(YKKAKT; 289), sasF (SRRNKL; 618), glcB (IRKFKL; 415), sasA (MHHTHS;1263), SAR0879 (LKKIKG; 573), SAR0986 (YRHLKP; 754), SAR1559 (IRKAHQ;206), SAR2393 (PKRKVVKI; 149), and sasG (VRKARS; 140); from methicillinsensitive Staphylococcus aureus, strain 476 (MSSA476): SAS2383 (VRKARS;140; and VKKSKI; 1319), SAS1682 (LKKNKY; 4; and IRKYKV; 11), SAS1063(YKKAKT; 282), SAS1657 (YHKAKT; 484), SAS2532 (SRRNKL; 626), SAS2540(MHHTHS; 2187), SAS0082 (LKKIKG; 573), SAS1011 (IRKAHQ; 206), SAS2035(PKRKVVKI; 149), and SAS2424 (IRKFKL; 415); and from Staphylococcusepidermidis, strain RP62A: SERP1316 (FRKQKF; 4; VHRLKV; 352; and IHKIKP;3234), SERP0660 (LKKWKV; 4; and IRRAHQ; 212), SERP0719 (TRKNHY; 13),SERP1482 (VKRFKN; 1730), SERP1654 (MKKSKV; 1), SERP2264 (MKRIKT; 393),SERP0207 (NRKNKN; 887), and SERP1691 (PKKIKN; 72).

In one embodiment, an antibody is generated against a linear heparinbinding motif which is conserved among MSSA, MRSA, and S. epidermidis.In one embodiment, the conserved heparin binding motif is selected fromthe group consisting of LKKNKY, LKKNKY, LKKWKV, LKKIKG, LKKIKG, VRKARS,VRKARS, YKKAKT, YKKAKT, YHKAKT, PKRKVVKI, PKRKWKI, IRKAHQ, IRKAHQ, andIRRAHQ.

Putative linear HBM were identified in the following cell wall orputative cell wall proteins from various yeast species, as shown inTable V where a check mark indicates that the motif is identical to themotif found in C. albicans, including C. dubliniensis (Int1 (YKKRFFKL),Eno1 (AKKGKF), and Tdh3 (GHKIKV) proteins), C. parapsilosis (Tdh3protein (GHKIKV)), C. tropicalis (Int1 (FKRRFFKL), and Tdh3 (GHKIKV)proteins), C. glabrata (Int1 (FKKRFFTL) protein), Lodderomyceselongisporus (Int1 (FKKFIFKL) and Tdh3 (GHKIKV) proteins), and A.nidulans (Int1 (FKKRFFKL) protein). In embodiments, an antibody directedto a region of these proteins containing the putative HBM or the regionof these proteins containing the putative HBM may be used in thedescribed methods.

TABLE I  Methicillin resistant Staphylococcus aureus, strain 252 MRSA252“Cell wall” “Cell wall” Have in in GO signaling Motif in StartGI Accession Name Description Description annotation peptide SequenceMotif Location G1:49484003 sasC putative surface anchored Yes Yes LPNTG2153 protein GI:49483291 isdB iron-regulated heme-iron Yes Yes LPQTG 616binding protein GI:49484843 sasF putative surface anchored Yes Yes LPKAG588 protein GI:49484739 glcB PTS system, glucose- Yes LPAAG 22specific IIABC component G1:49484850 sasA putative serine rich Yes LPDTG1308 repeat containing protein GI:49482351 SAR0879 putative myosin- YesLPKAG 57 crossreactive antigen GI:49482500 SAR0986 putative nitric oxideYes LPSAG 230 reductase GI:49483239 SAR1559 putative cobalt transportYes LPITG 258 protein GI:49484356 SAR2393 hypothetical protein Yes LPTAG177 sasG virulence associated cell wall protein Ortholog RP62AHave heparin in MSSA476 ortholog  Ortholog orthology binding #of heparinGI Accession MSSA476 has motif? in RP62A has motif: motif?binding motifs GI:49484003 SAS1682 Yes SERP1316 Yes Yes 2 GI:49483291SAS1063 Yes Yes 1 GI:49484843 SAS2532 Yes SERP2264 Yes Yes 1 GI:49484739SAS2424 Yes Yes 1 GI:49484850 SAS2540 Yes GI:57865710 No Yes 1GI:49482351 SAS0082 Yes GI:57866574 No Yes 1 GI:49482500 Yes 1GI:49483239 SAS1011 Yes SERP0660 Yes Yes 1 GI:49484356 SAS2035 YesSERP1739 Yes Yes 1 Yes 1 Motif 1 Motif GI Accession type Motif 1 seqafter Motif 2 type Motif 2 seq Motif lifter GI:49484003 Cardin LKKNKY 3Cardin IRKYKV 10 GI:49483291 Cardin YKKAKT 288 GI:49484843 Cardin SRRNKL617 GI:49484739 Cardin IRKFKL 414 GI:49484850 Cardin MHHTHS 1262GI:49482351 Cardin LKKIKG 572 GI:49482500 Cardin YRHLKP 753 GI:49483239Cardin IRKAHQ 205 GI:49484356 Wentraub PKRKVVKI 148 Cardin VRKARS 139found by MKH

TABLE II  Methicillin sensitiveie Staphylococcus aureus, strain 476MSSA476 “Cell wall” GI “Cell wall” in in GO  ID MSSA476 refSeq AccessionName Description Description annotation SAS2383 YP_044496.1 GI:49487275SAS2383 putative cell wall- Yes Yes anchored protein SAS1682 YP_043802.1GI:49486581 SAS1682 putative surface anchored protein SAS1063YP_043187.1 GI:49485966 SAS1063 iron-regulated heme- Yesiron binding protein SAS1657 YP_043776.1 GI:49486555 SAS1657haptoglobin-binding Yes surface anchored protein SAS2532 YP_044646.1GI:49487425 SAS2532 putative surface anchored protein SAS2540YP_044654.1 GI:49487433 SAS2540 putative cell wall- Yes Yesanchored protein SAS0082 YP_042206.1 GI:49484985 SAS0082putative myosin- crossreactive antigen SAS1011 YP_043135.1 GI:49485914SAS1011 putative cobalt transport protein SAS2035 YP_044146.1GI:49486925 SAS2035 hypothetical protein SAS2424 YP_044538.1 GI:49487317SAS2424 PTS system, glucose- specific IIABC component MRSA252 RP62A HaveHave Start Orthlog ortholog orthology heparin signaling Motif in Loca-in has Ortholog in has binding ID MSSA476 peptide Sequence Motif tionMRSA252 motif? RP62A motif? motif? SAS2383 Yes Yes LPKTG 1338 sasG aapYes Yes SAS1682 Yes Yes LPNTG 2150 sasC Yes SERP1316 Yes Yes SAS1063 YesYes LPQTG 609 isdB Yes Yes SAS1657 Yes Yes LPKTG 860 Yes SAS2532 Yes YesLPKAG 596 sasF Yes SERP2264 Yes Yes SAS2540 Yes LPDTG 2232 sasA YesGI:57865710 No Yes SAS0082 Yes LPKAG 57 SAR0879 Yes GI:57866574 No YesSAS1011 Yes LPITG 258 SAR1559 Yes SERP0660 Yes Yes SAS2035 Yes LPTAG 177SAR2393 Yes SERP1739 Yes Yes SAS2424 Yes LPAAG 22 glcB Yes Yes # ofheparin binding Motif  Motif Motif ID MSSA476 motifs 1 type Motif 1 seqafter 2 type Motif 2 seq Motif after SAS2383 2 Cardin VRKARS 139 CardinVKKSKI 1318 SAS1682 2 Cardin LKKNKY 3 Cardin IRKYKV   10 SAS1063 1Cardin YKKAKT 281 SAS1657 1 Cardin YHKAKT 483 SAS2532 1 Cardin SRRNKL625 SAS2540 1 Cardin MHHTHS 2186 SAS0082 1 Cardin LKKIKG 572 SAS1011 1Cardin IRKAHQ 205 SAS2035 1 Wentraub PKRKVVKI 148 SAS2424 1 CardinIRKFKL 414

TABLE III  Staphylococcus epidermidis, strain RP62A “Cell wall” in“Cell wall” in ID RP62A refSeq GI Accession Name Description DescriptionGO annotation SERP1316 YP_188888.1 GI:57867198 SERP1316cell wall surface anchor family protein Yes SERP0660 YP_188245.1GI:57866567 SERP0660 cobalt transport family protein SERP0719YP_188302.1 GI:57866639 SERP0719 cell wall surface anchor family proteinYes SERP1482 YP_189048.1 GI:57867352 SERP1482cell wall surface anchor family protein Yes SERP1654 YP_189219.1GI:57867536 SERP1654 cell wall surface anchor family protein YesSERP2264 YP_189815.1 GI:57865679 SERP2264cell wall surface anchor family protein Yes SERP0207 YP_187803.1GI:57866125 SERP0207 sdrG protein SERP1691 YP_189256.1 GI:57867615SERP1691 cell division protein, FtsW/RodA/SpoVE familyStrain RP62A (continuation) Have MSSA476 Ortholog  MRSA252 Have heparin# of heparin signaling Motif in Start Orthlog in ortholog in orthologybinding binding ID RP62A peptide Sequence Motif Location MSSA476has motif? MRSA252 has motif? motif? motifs SERP1316 Yes Yes LPEAG 3654SAS1682 Yes sasC Yes Yes 3 SERP0660 Yes LPITG 264 SAS1011 Yes SAR1559Yes Yes 2 SERP0719 Yes Yes LPETG 787 Yes 1 SERP1482 Yes Yes LPDTG 1937Yes 1 SERP1654 Yes Yes LPETG 165 Yes 1 SERP2264 Yes Yes LPATG 639SAS2532 Yes sasF Yes Yes 1 SERP0207 Yes LPDTG 852 SAS0521 Yes bbp YesYes 1 SERP1691 Yes LPITG 352 SAS1988 Yes SAR2371 Yes Yes 1Strain RP62A (continuation) ID RP62A Motif 1 type Motif 1 seqMotif after Motif 2 type Motif 2 seq Motif after Motif 3 typeMoitif 3 seq Motif after SERP1316 Cardin FRKQKF 3 Cardin VHRLKV 351Cardin IHKIKP 3233 SERP0660 Cardin LKKWKV 3 Cardin IRRAHQ 211 SERP0719Cardin TRKNHY 12 SERP1482 Cardin VKRFKN 1729 SERP1654 Cardin MKKSKV 0SERP2264 Cardin MKRIKT 392 SERP0207 Cardin NRKNKN 886 SERP1691 CardinPKKIKN 71

TABLE IV Linear Heparin Binding Motifs Conserved Among 2 or 3 Staphylococcus speciesMRSA sasC LKKNKY sar0879 LKKIKG sasG VRKARS isdB YKKAKT sar2892 MSSAsasC LKKNYY sas0082 LKKIKG sas2383 VRKARS sas1063 YKKAKT sas1657 YHKAKTsas2035 St. epi serp0660 LKKWKV MRSA PKRKWKI sari 559 IRKAHQ MSSAPKRKWKI sas1011 IRKAHQ St. epi serp0660 IRKAHQ

TABLE V Heparin Binding Motifs in various yeast species HEPARIN-BINDINGMOTIF Lodderomyces C. albicans XXXXXXX C. albicans C. dubliniensis C.parapsilosis C. tropicalis C. glabrata elongisporus A. nidulans MotifProtein FKKRFFKL Int1 √ YKKRFFKL no FKRRFFKL FKKRFFTL FKKFIFKL √ LRRLRTSsa2/Ssb1 √ no AKKGKF Eno1 √ √ no no no GHKIKV Tdh3 √ √ √ √ √ √identical

The interaction of C. albicans with heparin or heparin-like compoundsmay play a key role in clinical settings where heparin is used (e.g.central venous catheters, dialysis catheters) or possibly in tissueswhere heparan sulfates are preferentially expressed.

Because antibodies raised against a linear heparin binding motifinhibited binding of C. albicans to heparin, the heparin binding peptideHKQEKQKKHQIHKV (SEQ ID NO: 4) could be used as an immunizing antigen inimmunocompetent patients to elicit antibodies that protect againstheparin binding.

For example, a peptide from streptococcal M protein has been used tomake a vaccine for rheumatic fever (62). Alternatively, antibodiesraised against this peptide in immunized humans can be used for passiveimmunization. This technology is used with commercially availableantibodies such as hepatitis B immune globulin (HBIG) or botulism immuneglobulin (BabyBIG®) (63). Hepatitis B immune globulin is commerciallyavailable; botulism immune globulin is made and distributed by theCalifornia Department of Public Health and is FDA-approved. A humanizedmonoclonal antibody recognizing a desired heparin binding motif could bemade using technology for the production of palivizumab (Synagis®), ahumanized monoclonal antibody that is given monthly to prematurenewborns to prevent infection with respiratory syncytial virus (64).Because similar heparin binding motifs are also found in S. epidermidisand S. aureus, using heparin binding motifs as antigens is a first stepin developing vaccines against three of the most common causes ofcentral line-associated bloodstream infection.

Antibodies raised against a linear heparin binding motif expressed by asurface protein of C. albicans inhibited adhesion of the yeast toheparin in vitro and abolished biofilm formation in vivo. Similarheparin binding motifs occur in surface proteins from S. epidermidis andS. aureus, two organisms which are also major causes of biofilm-relatedinfections.

In one embodiment, an antibody reactive with a heparin binding motifexpressed on the surface of a microorganism is administered to anindividual. The microorganism may be, but is not limited to, a Candidaspecies or a Staphylococcus species. In one embodiment, themicroorganism is C. albicans, S. epidermidis and/or S. aureus. In oneembodiment, the heparin binding motif is identified by a computer-basedalgorithm analysis of protein sequences expressed by a microorganism. Inone embodiment, the heparin binding motif to which the antibody wasgenerated is derived from Int1. In one embodiment, the antibody isdirected to the peptide HKQEKQKKHQIHKV (SEQ ID NO: 4), or a fragment ofthis peptide. In one embodiment, the fragment is at least seven aminoacids of the peptide. In one embodiment, the surface expression of theheparin binding motif depends on the life-cycle stage of themicroorganism. In one embodiment, the antibody is a polyclonal antibody.In one embodiment, the antibody is a monoclonal antibody.

In one embodiment, a peptide that corresponds to a linear heparinbinding motif expressed on the surface of a microorganism, or a portionof the peptide, is provided. In one embodiment, the peptide is used togenerate antibodies directed against the peptide, using methods known inthe art. Alternatively, a peptide is provided combined with additionalcarriers or as a component of a complex, e.g., an adjuvant non-toxic tohumans. Such an adjuvant could be aluminum hydroxide used in Engerix-B®(commercially available hepatitis B vaccine), and in Comvax®(commercially available vaccine for Haemophilus influenzae type b andhepatitis B). The peptide corresponds to a linear heparin binding motif,or a portion of the motif, of a microorganism. In one embodiment, themicroorganism is a Candida species and/or a Staphylococcus species. Inone embodiment, the microorganism is C. albicans, S. epidermidis and/orS. aureus. In one embodiment, the heparin binding motif is identified bya computer-based algorithm analysis of protein sequences expressed by amicroorganism. In one embodiment, the heparin binding motif is derivedfrom Int1. In one embodiment, the peptide is HKQEKQKKHQIHKV (SEQ ID NO:4), or a fragment of SEQ ID NO: 4. In one embodiment, the peptide is afragment of the peptide HKQEKQKKHQIHKV (SEQ ID NO: 4), where thefragment contains at least seven amino acids of the peptide. In oneembodiment, the surface expression of the heparin binding motif dependson the life-cycle stage of the microorganism. In one embodiment, thepeptide is used to generate an antibody using methods known in the art.

In one embodiment, a method is provided for ameliorating biofilmformation on a surface of an implanted device in a patient. In oneembodiment, the method comprises administering an antibody to a patient,and ameliorating biofilm formation on a surface of an implanted devicein a patient, wherein the antibody is directed to a heparin bindingmotif expressed on the surface of a microorganism. In one embodiment,the antibody is administered intravenously at a dose not more than 2grams/kg given as an infusion over 10-24 hours. Since the half-life ofantibody preparations is about four weeks, antibody administration wouldbe repeated at monthly intervals over the life of the catheter. In oneembodiment, the antibody has been modified to increase safety and/orefficacy in a human patient. In one embodiment, the antibody has beenhumanized using methods known in the art. In one embodiment, thedescribed antibody is provided as a pharmaceutical composition with atleast one biocompatible excipient, e.g., buffers, preservatives,tonicity adjusting agents, pH adjusting agents, osmolality adjustingagents, etc, as known to one skilled in the art. Without being held to asingle theory, the antibody blocks binding between a heparin bindingmotif-containing protein expressed on the surface of a microorganism andheparin or heparin sulfate, which is expressed by human cells lining thelumen of the implanted device (FIG. 10). Binding of the antibody to themicroorganism prevented its attachment to heparin or to heparin sulfatemoieties. In the rat model of Candida infection of central venouscatheters, the disclosed antibody recognizing a peptide encompassingMotif 1 in Candida albicans Int1 (also called anti-KKHQ antibody (“KKHQ”disclosed as SEQ ID NO: 6)) showed excellent in vivo results. Theantibody recognizing Motif 1 (anti-KKHQ antibody (“KKHQ” disclosed asSEQ ID NO: 6)) substantially inhibited biofilm production, as shown inFIG. 9. FIGS. 11 and 12A-C demonstrate the anti-KKHQ antibody iseffective against various Candida species.

The administration of the described antibody is referred to as passiveimmunization.

In one embodiment, the method comprises administering a peptide to apatient, where the peptide encompasses a heparin binding motif, or afragment of the motif, that is expressed on the surface of amicroorganism. The peptide is 14 amino acids in length, with amino acidsboth preceding and following the 8 amino acids of the heparin bindingmotif. In one embodiment, the peptide is HKQEKQKKHQIHKV (SEQ ID NO: 4),or a fragment thereof. In one embodiment, the described peptide isprovided as a pharmaceutical composition comprising at least onebiocompatible excipient including but not limited to buffers,stabilizing agents, solubility enhancing agents, tonicity agents, etc.as known to one skilled in the art.

Without being held to a single theory, the peptide serves as an antigento immunize the patient. It provides immunity by promoting endogenousgeneration of antibodies directed to the peptide, where theendogenously-produced antibodies block binding between heparin, which isadministered through the catheter or is bound to a surface of animplanted medical device, and a heparin binding motif-containing proteinexpressed on a microorganism surface. Administration of the describedpeptide is referred to as active immunization. Peptide vaccines aretypically administered subcutaneously or intramuscularly. Immunizingdose of the peptide and frequency of immunization are experimentallydetermined with humans.

The patient being treated may be naturally immunodeficient by having anunderdeveloped immune response, e.g., a premature newborn. The patientbeing treated may have a normal immune response, but may beimmunocompromised due to particular circumstances, e.g., as a result oftreatment such as chemotherapy, or disease, or burns. In one embodiment,the disclosed antibody is administered to an immunodeficient patient. Inone embodiment, the disclosed peptide is administered to annon-immunodeficient patient, e.g., an otherwise well adult or child whowill receive long term antibiotics through a central line for thetreatment of a serious infection, e.g., endocarditis.

In one embodiment, the implanted medical device, e.g., catheter, centralline, hemodialysis catheter, peritoneal catheter, PICC line, plasticcatheters such as central venous catheters, urinary tract catheters,central nervous system shunt catheters, peritoneal dialysis catheters,dialysis shunts, etc., is implanted in a vein, artery, or a body cavity.In one embodiment, the implanted medical device is a plastic device. Inone embodiment, the implanted device is present in the patient's bodyfor a period of time ranging from minutes to six weeks or longer. In oneembodiment, the above described methods may be performed before thedevice is implanted in the patient. In one embodiment, a first dose ofthe antibody is administered before the medical device is implanted. Inthis embodiment, a peripheral intravenous line could administer thisfirst dose of the antibody if the device to be implanted is a catheter.

In one embodiment, the described methods decrease the virulence of amicroorganism, i.e., the method decrease the ability of a microorganismto cause infection in a patient.

All references cited are expressly incorporated by reference herein intheir entirety.

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The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on Mar. 8, 2013, isnamed 070248_(—)94_SL.txt and is 30,824 bytes in size.

The embodiments described in the specification are only specificembodiments of the inventors who are skilled in the art and are notlimiting. Therefore, various changes, modifications, or alterations tothose embodiments may be made without departing from the spirit of theinvention or the scope of the following claims.

What is claimed is:
 1. A composition comprising at least onebiocompatible excipient and an antibody reactive with peptideHKQEKQKKHQIHKV (SEQ ID NO: 4) or a fragment of at least seven aminoacids of SEQ ID NO:
 4. 2. A composition comprising at least onebiocompatible excipient and an antibody reactive with a heparin bindingmotif expressed on the surface of a microorganism.
 3. The composition ofclaim 2 where the microorganism is a Candida species or a Staphylococcusspecies.
 4. A composition comprising an isolated peptide HKQEKQKKHQIHKV(SEQ ID NO: 4) or a fragment of at least seven amino acids of SEQ ID NO:4.
 5. The composition of claim 4 where the peptide is a heparin bindingmotif, or a portion thereof, that is expressed on the surface of amicroorganism.
 6. The composition of claim 5 where the microorganism isa Candida species or a Staphylococcus species.
 7. The composition ofclaim 1 where the peptide is derived from Int1 protein.
 8. Thecomposition of claim 5 where the surface expression of the heparinbinding motif depends on the life-cycle stage of the microorganism. 9.The composition of claim 1 where the antibody is a polyclonal antibody.10. The composition of claim 1 where the antibody is a monoclonalantibody.
 11. A method to reduce or prevent biofilm on a surface of anindwelling or implanted medical device in a patient, the methodcomprising administering before, during, and/or after installation orimplantation of a medical device in the patient a composition comprisingat least one biocompatible excipient and an antibody reactive with apeptide HKQEKQKKHQIHKV (SEQ ID NO: 4) or a fragment of at least sevenamino acids of SEQ ID NO: 4 under conditions to result in reducedheparin-binding to a surface of the implanted medical device, resultingin reduced or prevented biofilm formation on the medical device.
 12. Themethod of claim 11 where, when the composition comprises the peptide,the peptide antigenically immunizes the patient by endogenouslygenerating anti-peptide antibodies that reduce or prevent biofilmformation on the surface of the indwelling or implanted device byreducing or preventing microorganism heparin-mediated-binding to thedevice surface.
 13. The method of claim 11 where the implanted device isselected from the group consisting of a venous catheter, an arterialcatheter, a central line, a hemodialysis catheter, a peritonealcatheter, a peripherally inserted central catheter, a urinary tractcatheter, a central nervous system shunt, a peritoneal dialysiscatheter, a dialysis shunt, and combinations thereof.
 14. The method ofclaim 11 resulting in decreased patient infection.
 15. The method ofclaim 11 resulting in decreased microorganism virulence.
 16. A kitcomprising instructions for administering a composition to a patientcandidate for medical device installation or implantation, and at leastone of a composition comprising at least one biocompatible excipient andan antibody reactive with a peptide HKQEKQKKHQIHKV (SEQ ID NO: 4) or afragment of at least seven amino acids of SEQ ID NO: 4, or a compositioncomprising a peptide HKQEKQKKHQIHKV (SEQ ID NO: 4) or a fragment of atleast seven amino acids of SEQ ID NO:
 4. 17. An antibody reactive withpeptide HKQEKQKKHQIHKV (SEQ ID NO: 4) or a fragment of at least sevenamino acids of SEQ ID NO:
 4. 18. An antibody reactive with peptideQKKHQIHK (SEQ ID NO: 1), THKGRF (SEQ ID NO: 40), MKRGKP (SEQ ID NO: 48),FKKRFFKL (SEQ ID NO: 2), or SHKTRA (SEQ ID NO: 3).